Optogenetic Stimulation and Cell Localization for Three-Dimensional Cellular Networks

• Applicant: Professor Dr.-Ing. Jürgen W. Czarske
• Subject Area: Biomedical Systems Technology ; Measurement Systems
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 524497646

This project aims to realize and investigate a laser-optical system for optogenetic stimulation and microscopic analysis of three-dimensional cell networks using cardiomyocytes as a case study. The pumping function of the heart is ensured by the electrical coupling of the cardiomyocytes. The contraction movement of all cells is synchronized by macroscopic action potential waves in the form of spatio-temporal patterns. Cardiac arrhythmias are mainly due to disturbances in the propagation of the excitation waves, and their occurrence as well as their tissue-sparing termination are still the subject of research and development. At this point, optogenetics, which deals with the control of the activity of transgenic cells by means of light, has introduced a paradigm shift: Single cells or whole groups of cells can be activated by light stimulation in a cell-specific manner with millisecond time resolution and with cellular spatial resolution. By means of in vitro experiments on artificial cardiomyocyte networks, perturbations of excitation conduction can for the first time be specifically induced, observed, controlled, and approaches to terminate them derived, and thus spiral waves can for the first time be fundamentally better studied and phenomenologically understood. However, previous studies have been performed only on cardiac myocyte monolayers and thus reduced to a 2D problem. The transfer to multilayer, three-dimensional cell samples faces challenges such as sample-induced aberrations and light scattering. These exist both in light stimulation and in microscopic observation of cell response. In this project, the aforementioned challenges will be addressed by spatial and temporal light field shaping. A nonlinear two-photon process with ultrashort laser pulses will be used to reduce the influence of light scattering during excitation. At the same time, the depth selectivity of the excitation will be improved compared to the single-photon process by selectively controlling the spectral laser pulse components. It will be demonstrated for the first time how this can be used to generate complex spatiotemporal stimulation patterns even in the bulk. A further increase in penetration depth will be pursued by implementing an adaptive optical wavefront correction to correct for system-inherent as well as sample-induced aberrations. Microscopic observation and localization of the cell reaction will be achieved for the first time in three dimensions using a double-helical point spread function in a single-shot image. The project will pave the way for future in vitro optogenetic experiments on three-dimensional samples such as multilayer cell assemblies and complex organoids as organ-like microstructures that can be used to model and study diseases.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Lars Büttner